The overall scientific mission of the Cartilage Biology and Orthopaedics Branch is the study of the biology of cartilage tissues and the application of such knowledge to musculoskeletal and orthopaedic medicine. This project focuses on cartilage development, functional cartilage tissue engineering, the cellular basis of orthopaedic implant stability, physical influences on skeletal tissue function, and the biology of skeletal tissue injury repair. There are five interrelated parts to the project: (1) Cellular and Molecular Mechanisms of Chondrogenesis. Using embryonic skeletal mesenchymal cells and multipotent embryonic mesenchymal cell lines, we have analyzed the role of cell-cell and cell-matrix interactions, growth factors and other signaling molecules, and gene expression and regulation events during chondrogenesis. Topics include: a) N-cadherin mediated cell adhesion; b) gap junction communication; c) cell interaction with fibronectin isoforms; d) members of the transforming growth factor-beta (TGF-beta) superfamily, i.e. bone morphogenetic proteins (BMPs) and Growth/Differentiation Factor 5 (GDF5); e) Wnt family of signaling proteins; f) transcription factors, such as cbfa1, AP1, paraxis, scleraxis, and other basic helix-loop-helix proteins; and g) mechanism of action of environmentally derived agents that cause structural malformations, such as carbon monoxide. Results from these studies point to the initial cellular condensation event, and the cross-talk of multiple signaling pathways, as key regulatory events of developmental chondrogenesis. (2) Mesenchymal Stem Cell Biology and Cell-Based Cartilage Tissue Engineering. The information gained from these developmental studies are also being applied to the system of adult tissue derived mesenchymal stem cells (MSCs) for the purpose of cartilage tissue engineering. Multipotent MSCs are isolated from bone marrow stroma and from adult trabecular bone and studied in vitro for their ability to undergo multi-lineage differentiation along the osteogenic, chondrogenic, and adipogenic pathways. The mechanisms of action of growth factors (e.g. TGF-beta superfamily members), signaling molecules (e.g. Wnts), and hormonal regulators (e.g. glucocorticoids) in the mainteance of their undifferentiated state and lineage commitment are being analyzed. Novel methods have been developed for efficient gene transduction, using electrical field based and nucleofection protocols, to modulate the expression of these key factors in mesenchymal stem cells and to examine the effects on cellular differentiation. In addition to establishing protocols to optimize MSC isolation and enrichment, we are developing new methods to construct three-dimensional biodegradable scaffolds to seed MSCs under chondrogenic conditions for cartilage tissue engineering applications. Two approaches have been successful: a) fabrication of single-unit, osteochondral constructs suitable as implants for cartilage re-surfacing; and b) development of novel three-dimensional nanofibrous scaffolds consisting of biodegradable polymers produced by electrospinning. Specifically, we are custom-designing biomaterial scaffolds to assemble composite tissues of complex architecture. We believe that the knowledge gained from the study of developmental chondrogenesis will be important in designing approaches to modulate the cellular and molecular activities of chondrogenic MSCs in three-dimensional cartilage tissue engineering. Elucidating the biological characteristics of MSCs and how they respond to environmental signals is therefore fundamental to the advancement of tissue engineering. We are currently developing clonal MSC cell lines harboring differentiation-specific marker gene constructs as read-out cell systems, e.g. for functional gene cloning applications, as well as gene microarray approaches to profile gene expression profiles during differentiation to identify key regulatory signals. Particularly noteworthy is our recent finding that MSCs possess transdifferentiation potential, underscoring the possibility of ?stemness? regulatory genes. (3) Cellular Mechanism of Wear Debris Mediated Osteolysis. Finally, our recent studies have also addressed the cellular mechanisms responsible for implant wear debris mediated osteolysis, which is primarily responsible for aseptic implant loosening. Specifically, our results indicate that the presence of titanium particulate debris suppresses osteogenic differentiation and enhances apoptosis in cultures of MSCs in vitro, both of these responses likely contributing to compromised periprosthetic osteogenic tissue response and implant loosening. The identity and involvement of specific cytokines and signaling pathways in the affected cellular events are being investigated. (4) Analysis of Physical Influences on Skeletal Biology. Skeletal tissues are uniquely adapted to responding to mechanical influences. We are currently designing mechanoactive bioreactor systems and using MSC-based tissue constructs to analyze the cellular and molecular basis of the biological responses. By varying the nature of the biomaterial scaffold, the mixture of cell types, and the growth factor treatment, we aim to decipher the crosstalk among various signal transduction pathways. We are also applying a mechanoactive bioreactor system for the tendon/ligament tissue engineering. (5) Animal Models of Skeletal Injury Repair and Regeneration. Three models are being investigated: a) the role of GDF5 in mouse tibial fracture repair, b) cellular and molecular analysis of distraction osteogenesis in a mouse model, and c) the pathogenesis of osteoarthritis in a supraphysiological impact-induced cartilage degeneration rabbit model.